"Cross-Pollination Leads to Triple Herbicide Resistance" - Syngamy

ISB News Report
March 2001

Source

Hall L, Topinka K, Huffman J, Davis L, and Good A. 2000. Pollen flow between herbicide-resistant Brassica napus is the cause of multiple-resistant B. napus volunteers. Weed Science 48: 688-694.

Jim Westwood
Department of Plant Pathology, Physiology, and Weed Science
Virginia Tech

One of the risks frequently cited in association with transgenic crops is the escape of a foreign gene via sexual reproduction. The recipient plant in such cases may be a non-transgenic variety of the same crop or a sexually compatible relative. Depending on the gene and trait considered, adverse environmental or agricultural impacts may result from such transfers, ranging from issues of genetic purity of neighboring crops to the generation of "super weeds." While this issue is receiving increasing attention by researchers, a recent report by Hall et al.(1) describes a truly remarkable example of herbicide resistance transfer via pollen among Brassica napus varieties. What is unusual here is not so much that it happened at all, but that it occurred rapidly and multiple times, such that, through completely random crossing, certain plants were found to be resistant to three different herbicides.

The Problem

The events that precipitated this study occurred in Alberta, Canada where in 1997 a producer planted three varieties of B. napus in the same or adjacent fields. One variety was resistant to glyphosate, another was resistant to glufosinate, and a third resistant to herbicides of the imidazolinone type. In field 1, the producer started planting glufosinate-resistant B. napus, but, after 15 hectares had been sowed, switched to imidazolinone-resistant B. napus for the remainder of the field. In field 2, which was located across a road (22 m from the edge of field 1 containing the glufosinate-resistant plants), the producer planted glyphosate-resistant B. napus. The next year, the producer fallowed field 1 and planted part of field 2 with imidazolinone-resistant B. napus. Weeds in the fallowed field 1 were sprayed with glyphosate, but the producer noticed that B. napus volunteers in this field were not being controlled by the herbicide.

At this point, Hall and company stepped in and surveyed field 1 to determine whether herbicide resistance had developed. They collected B. napus volunteers that had survived the glyphosate treatments and grew them in the greenhouse in order to obtain seeds for further study. The resulting generation was then tested for resistance to glyphosate by spraying seedlings with the herbicide. They found that 20 of 34 individuals tested had a high percentage of offspring showing resistance to glyphosate. This result could be accounted for by either of two mechanisms: 1) direct movement of glyphosate-resistant seeds from field 2 across the road to field 1 (most likely transported by farm equipment), or 2) movement of pollen such that cross-hybridizations occurred between the glyphosate-resistant variety and either the glufosinate- or imidazolinone-resistant varieties. Although mechanical operations (planting and harvesting) in the two different fields were not performed at the same time, the direct movement of seed from field 2 to field 1 remained a possibility.

Evaluation of Glyphosate-resistant Volunteers

To confirm whether pollen flow was responsible for the glyphosate resistance, Hall et al. set forth three tests. First, if the volunteer plants had gained glyphosate resistance through pollen flow, this would have been conferred by the male parent, while glufosinate or imidazolinone resistance would have been inherited from the maternal plant. Thus, they would be resistant to two herbicides rather than just one. Second, if the resistance trait had been transferred via pollen, the progeny of the resistant plants would show a pattern of segregation for resistance consistent with Mendelian ratios for a single-locus dominant trait. Third, RFLP analysis of the progeny using markers specific for each of the parental lines would indicate whether the volunteer plants were products of a hybridization of two lines. Eight volunteer B. napus plants were put through these tests and seven passed them all, demonstrating that gene flow via pollen was the primary mechanism of resistance transfer.

Progeny of additional plants were characterized solely on the basis of resistance to herbicides. In the section of field 1 originally planted with glufosinate-resistant B. napus, all nine plants tested had progeny resistant to both glyphosate and glufosinate. In the section planted with imidazolinone-resistant plants, the progeny of 10 of 11 plants were resistant to both glyphosate and the imidazolinone herbicide, imazethapyr (the lone exception to this being a glufosinate-resistant individual that was likely moved as a seed from the nearby glufosinate resistant section of the field).

Triple Resistance

In addition to the cases of double resistance, two plants from field 1 gave rise to progeny resistant to all three herbicides. This was attributed to sequential hybridization among the plants. In one such case it is probable that a glufosinate-resistant plant was pollinated by a glyphosate-resistant plant in 1997. The following year, a progeny of this plant was selected by application of glyphosate to kill competing vegetation and was subsequently cross-pollinated by imidazolinone-resistant B. napus planted in field 2 in 1998.

The triple resistance in the second plant is proposed to have arisen by a different sequence of events, with the first cross occurring between glufosinate- and imidazolinone-resistant plants. A progeny of this cross is thought to have escaped glyphosate treatment and crossed with one of the glyphosate-resistant volunteers. Although the end result is the same, this illustrates how all possible combinations and sequences of events are possible.

Lessons From This Situation

This report demonstrates that pollen flow among individuals of an outcrossing species can be a very effective method for transmitting genes. B. napus is capable of both selfing and outcrossing, having an outcrossing frequency of 20-30%. In the field, the actual cross-hybridization rate is a function of distance, with percent outcrossing diminishing the farther the recipient is from the pollen source. It is therefore interesting that one of the triple-resistant plants was found over 550 m from the pollen sources, greatly exceeding the 100-m buffer mandated for seed producers. Also, in cases such as this field situation where large numbers of plants are involved, even a low percentage of outcrossing can result in significant transfer of genes via pollen. It is important to note that this research is dealing with intraspecific hybridization. Hybridization between Brassica crops and several wild relatives has been reported, but may occur at lower frequencies and hybrid plants may suffer from lack of vigor or fertility.

The authors point out that the circumstances that gave rise to the triple-resistant B. napus are highly unusual in that three varieties harboring different herbicide-resistance genes were planted in close proximity in the same year. Then the next year the only weed control method used in the fallowed field 1 was the herbicide glyphosate. B. napus is susceptible to many weed control measures, so in the second year the glyphosate-resistant volunteers should properly have been controlled by herbicides with different modes of action, tillage operations, or cultural practices. Fortunately, seeds of B. napus have a relatively short persistence (duration of dormancy of four to five years in the soil) so proper management practices will eliminate multiple-resistant weed seeds in a relatively short period of time. Nevertheless, this report points out just how rapidly genes can move within an outcrossing crop and why planting distance and crop rotation precautions and herbicide/weed control techniques need to be varied regularly to avoid developing problematic volunteer weeds. (For more information, see "Outcrossing Between Canola Varieties - A Volunteer Canola Control Issue") This example should serve as a warning to producers to use their new herbicide-resistant crops wisely according to guidelines.

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